Vaginal HIV microbicides offer great promise to reduce HIV transmission, but phase 3 microbicide trials have failed. In some studies, patients using the microbicides had higher HIV transmission rates than did subjects using placebos. There is no clear explanation for these failures, but one hypothesis holds that microbicides alter the vaginal microbial flora in ways that increase inflammation or activate potential HIV host cells, enhancing transmission. Studies examining the effects of microbicides on the vaginal flora found few significant effects on the microbiome, but they used conventional culture techniques. Recent studies using molecular, culture-independent techniques showed that the flora in many human microbial environments, including the vagina, is much more complex than previously appreciated and that conventional culture techniques only detect a small fraction of the microbes in the environment. We propose to use these new culture-independent techniques to explore the hypothesis that microbicides alter the vaginal microbiome in ways that can potentially enhance HIV transmission via these specific aims: 1) Examine the vaginal microbial flora before and after microbicide application in a CONRAD repeat phase 1 study of nonoxynol-9 (N-9), cellulose sulfate (CS), and placebo using Affymetrix PhyloChip microarrays 2) Examine the portfolio of expressed genes in the vaginal microbiome before and after microbicide application using microbial cDNA sequencing in the phase 1 study, and 3) Examine the microbial species composition before and after microbicide application in the CONRAD CS phase 3 study that failed using the PhyloChip and direct 16S rRNA gene sequencing. The main milestone we propose to transition from the initial R21 phase of the project to the R33 phase is the demonstration that microbicide use leads to a significant alteration in the vaginal flora as assessed by the PhyloChip. Determining whether microbicide application is associated with vaginal microbiome changes that could enhance HIV transmission would aid understanding of the failure of the previous phase 3 trials and would help future microbicide development efforts because, if harmful changes in vaginal flora are associated with microbicide use, future microbicide development efforts would require careful measures to avoid inducing potentially harmful changes in the vaginal microbiome. Vaginal microbicides for the prevention of HIV sexual transmission offer great theoretical promise to reduce HIV sexual transmission and blunt the HIV pandemic, particularly in regions with the highest HIV prevalence rates. Unfortunately, several large late phase trials of HIV microbicides have failed for unknown reasons, with the research subjects using the microbicides having rates of HIV transmission higher than subjects using placebos. We hypothesize that one factor contributing to the failure of the microbicides is that their use produces a harmful change in the microbial flora living in the vagina, which leads to inflammation or activation of the cells that HIV replicates in, increasing the risks of HIV transmission. In our study, we propose to use new molecular biological techniques to comprehensively catalog essentially all of the microbes living in the vagina and determine how the use of HIV microbicides alters the population of the microbes. Determining that the use of HIV microbicides lead to a significant, potentially harmful alteration in the population of vaginal flora would help explain the failure of the existing microbicides to prevent HIV transmission and may help enable the development of new, more effect HIV microbicides.